1. Field of the Invention
The present invention relates to a process for preparing a prodrug formulation of ganciclovir and its pharmaceutically acceptable salts. More specifically, the invention relates to a process for preparing the L-monovaline ester derived from 2-(2-amino-1,6-dihydro-6-oxo-purin-9-yl)methoxy-1,3-propanediol and its pharmaceutically acceptable salts. The invention also relates to novel intermediates useful in the above process and to a process for preparing the intermediates.
2. Background Information
British Patent 1 523 865 describes antiviral purine derivatives with an acyclic chain in the 9-position. Among those derivatives 2-(2-amino-1,6-dihydro-6-oxo-purin-9-yl)methoxy-ethanol with the INN name acyclovir has been found to have good activity against herpes viruses such as herpes simplex.
U.S. Pat. No. 4,355,032 discloses the compound 9-[(2-hydroxy-1-hydroxymethylethoxy)methyl]guanine or 2-(2-amino-1,6-dihydro-6-oxo-purin-9-yl) methoxy-1,3-propanediol with the INN name ganciclovir. Ganciclovir is highly efficacious against viruses of the herpes family, for example, against herpes simplex and cytomegalovirus.
British Patent Application GB 2 122 618 discloses derivatives of 9-(2-hydroxyethoxymethyl)guanine of the generic formula: 
wherein X represents an oxygen or sulfur atom, R1 represents a hydroxy or an amino group, R2 represents a hydrogen atom or a group of the formula CH2OR3a and R3 and R3a may be the same or different, each represents an amino acid acyl radical and physiologically acceptable salts thereof. These compounds can be prepared by condensing a guanine derivative with a side chain intermediate in a strong polar solvent such as dimethylformamide or hexamethylphosphoramide, advantageously in the presence of a base, or by thermal condensation in the presence of a strong acid. These compounds are useful for the treatment of viral infections and have high water solubility which renders them of value in the formulation of aqueous pharmaceutical preparations. While the generic formula in the British patent application includes compounds in which R2 is the group xe2x80x94CH2OR3a, specific compounds of this group are not disclosed.
European Patent Application 0 375 329 A2 discloses prodrug compounds with the following formula 
wherein R and R1 are independently selected from hydrogen and an amino acid acyl residue providing at least one of R and R1 represents an amino acid acyl residue and B represents a group of the formulae 
in which R2 represents a C1-6 straight chain, C3-6 branched chain or C3-6 cyclic alkoxy group, or a hydroxy or amino group or a hydrogen atom and the physiologically acceptable salts thereof. These prodrug compounds are described as having advantageous bioavailability when administered by the oral route, resulting in high levels of the parent compound in the body.
Example 3(b) of European Patent Application 0 375 329 discloses the preparation of the bis(L-isoleucinate) ester of ganciclovir as a white foam. Example 4(b) discloses the preparation of the bis(glycinate) ester of ganciclovir as a white solid. Example 5(b) discloses the preparation of the bis(L-valinate) ester of ganciclovir as a solid. Example 6(b) discloses the preparation of the bis(L-alaninate) ester of ganciclovir as a syrup containing 90% of the bis-ester and 10% of the monoester. The bis-esters are prepared by reacting ganciclovir with an optionally protected amino acid or functional equivalent thereof; the reaction may be carried out in a conventional manner, for example in a solvent such as pyridine, dimethylformamide, etc., in the presence of a coupling agent such as 1,3-dicyclohexylcarbodiimide, optionally in the presence of a catalytic base such as 4-dimethylaminopyridine. The described bis-esters are non-crystalline materials which are difficult to process for the manufacture of oral pharmaceutical dosage forms.
British Patent Application No. 8829571 is the priority patent application for European Patent Application 0 375 329 and U.S. Pat. No. 5,043,339, and discloses amino acid esters of the compounds of the formula 
(wherein R represents a hydroxy or amino group or a hydrogen atom) and the physiologically acceptable salts thereof. Examples of preferred amino acids include aliphatic acids, e.g., containing up to 6 carbon atoms such as glycine, alanine, valine and isoleucine. The amino acid esters include both mono and diesters. The preparation of the diesters is identical to the preparation in European Patent Application 0 375 329; however, this patent application as well as European Patent Application 0 375 329 and U.S. Pat. No. 5,043,339 do not disclose the preparation of monoesters, or any data suggesting their usefulness.
Leon Colla et al., J. Med. Chem. (1983) 26, 602-604 disclose several water-soluble ester derivatives of acyclovir and their salts as prodrugs of acyclovir. The authors indicate that acyclovir cannot be given as eye drops or intramuscular injections because of its limited solubility in water and have therefore synthesized derivatives of acyclovir which are more water soluble than the parent compound. The authors disclose the hydrochloride salt of the glycyl ester, the hydrochloride salt of the alanyl ester, the hydrochloride salt of the beta-alanyl ester, the sodium salt of the succinyl ester, and the azidoacetate ester. The alanyl esters were prepared by conventional esterification methods, including reacting acyclovir with the corresponding N-carboxy-protected amino acid in pyridine, in the presence of 1,3-dicyclohexylcarbodiimide and a catalytic amount of p-toluenesulfonic acid and subsequent catalytic hydrogenation to give the alpha- and beta-alanyl esters as their hydrochloride salts.
L. M. Beauchamp et al., Antiviral Chemistry and Chemotherapy (1992), 3(3), 157-164 disclose eighteen amino acid esters of the antiherpetic drug acyclovir and their efficiencies as prodrugs of acyclovir, evaluated in rats by measuring the urinary recovery of acyclovir. Ten prodrugs produced greater amounts of the parent drug in the urine than acyclovir itself: the glycyl, D,L-alanyl, L-alanyl, L-2-aminobutyrate, D,L-valyl, L-valyl, DL-isoleucyl, L-isoleucyl, L-methionyl, and L-prolyl ester. According to the authors the L-valyl ester of acyclovir was the best prodrug of the esters investigated. These esters were prepared by methods similar to those employed by Colla et al.
European Patent Application 0 308 065 A2 discloses the valine and isoleucine esters of acyclovir, preferably in the L-form, as showing a large increase in absorption from the gut after oral administration, when compared with other esters and acyclovir. The amino acid esters are prepared by conventional esterification methods, including reacting acyclovir with an N-carboxy-protected amino acid or an acid halide or acid anhydride of the amino acid, in a solvent such as pyridine or dimethylformamide, optionally in the presence of a catalytic base. The amino acid esters of acyclovir may also be prepared by condensing a guanine derivative with an amino acid side-chain chain intermediate in a manner analogous to that disclosed in British Patent Application GB 2 122 618, discussed above.
PCT Patent Application WO 94/29311 discloses a process for the preparation of amino acid esters of a nucleoside analogue, including acyclovir and ganciclovir. This process comprises reacting a nucleoside analogue having an esterifiable hydroxy group in its linear or cyclic ether moiety, with a 2-oxa-4-aza-cycloalkane-1,3-dione of the formula 
wherein R1 may represent hydrogen, C1-4 alkyl or alkenyl group or other amino acid side chains, and R2 may represent hydrogen or a group COOR3 where R3 is a benzyl, t-butyl, fluorenylmethyl or an optionally halo substituted linear or branched C1-8 alkyl group. Preferred R1 groups include hydrogen, methyl, isopropyl and isobutyl, yielding respectively the glycine, alanine, valine and isoleucine esters of acyclovir or ganciclovir. Examples 1-3 of PCT Patent Application WO 94/29311 discloses only the condensation of acyclovir with the valine-substituted 2-oxa-4-aza-cycloalkane-1,3-dione (Z-valine-N-carboxyanhydride) by conventional procedures. While the amino acid esters of the PCT application include both the acyclovir and ganciclovir (DHPG) esters, the application does not disclose how to prepare the ganciclovir esters, much less the mono-esters of ganciclovir.
The L-monovaline ester derived from 2-(2-amino-1,6-dihydro-6-oxo-purin-9-yl)methoxy-1,3-propanediol and its pharmaceutically acceptable salts are potent antiviral agents and are described in European Patent Application 0 694 547 A. These compounds have been found to have improved oral absorption and low toxicity. This patent application also discloses certain processes for preparing these esters, different from those described herein.
The present invention relates to an improved process and novel intermediates whereby an acid addition salt of a mono-hydroxy protected ganciclovir is formed as a novel intermediate, which reduces impurities in the mono-valine ester end-product, compared to known intermediates. This also eliminates the costly and time consuming purification steps and allows the use of starting materials of lower purity, which, in turn, reduces overall production costs.
In a first aspect, this invention provides a process for preparing the compound of the formula (I): 
and pharmaceutically acceptable salts thereof, which compound is named hereinafter 2-(2-amino-1,6-dihydro-6-oxo-purin-9-yl)methoxy-3-hydroxy-1-propyl-L-valinate or mono-L-valine valine ganciclovir.
This process involves the condensation of a silylated guanine compound with a substituted glycerol derivative, optionally followed by formation of an acid addition salt of a mono-hydroxy protected ganciclovir as an intermediate; esterification of this product with an L-valine derivative and the removal of any protecting groups forms the prodrug of Formula (I). Optionally, the process can also include the formation of salts of the prodrug of Formula (I), the conversion of an acid addition salt of the prodrug of Formula (I) into a non-salt form, the optical resolution of a prodrug of Formula (I) or the preparation of the prodrugs of Formula (I) in crystalline form. Details of the process are described below.
In a second aspect, this invention provides compounds of Formula (IV) and Formula (V) which are useful intermediates for preparing mono-L-valine ganciclovir and its pharmaceutically acceptable salts.
The compounds of Formula (IV) are: 
wherein Z1 is hydrogen or an amino-protecting group, Y1 is halo, lower acyloxy or aralkyloxy, and Y2 is lower acyloxy.
The compounds of Formula (V) are: 
wherein Z1 is hydrogen or an amino-protecting group, Y2 is halo, lower acyloxy or aralkyloxy. Compounds of Formula (V) may optionally be converted into an acid addition salt; preferred is the hydrochloride salt.
A third aspect of this invention is a process for preparing the novel intermediates of Formula (IV) and (V).
Unless otherwise stated, the following terms used in the specification and claims have the meanings given below:
xe2x80x9cBOCxe2x80x9d means t-butoxycarbonyl.
xe2x80x9cCBZxe2x80x9d means carbobenzyloxy (benzyloxycarbonyl).
xe2x80x9cFMOCxe2x80x9d means N-(9-fluorenylmethoxycarbonyl).
xe2x80x9cDHPGxe2x80x9d means 9-[(1,3-dihydroxy-2-propoxy)methyl]guanine.
xe2x80x9cAlkylxe2x80x9d means a straight or branched saturated hydrocarbon radical having from 1-12 or one to the number of carbon atoms designated. For example, C1-7 alkyl is alkyl having at least one but no more than seven carbon atoms, e.g. methyl, ethyl, i-propyl, n-propyl, n-butyl, n-pentyl, n-heptyl and the like.
xe2x80x9cLower alkylxe2x80x9d means an alkyl of one to six carbon atoms.
xe2x80x9cArylxe2x80x9d means an organic radical derived from an aromatic hydrocarbon by the removal of one hydrogen atom. Preferred aryl radicals are aromatic carbocyclic radicals having a single ring (e.g., phenyl) or two condensed rings (e.g. naphthyl).
xe2x80x9cAralkylxe2x80x9d means an alkyl group in which an alkyl hydrogen atom is replaced by an above-defined aryl group.
xe2x80x9cAcylxe2x80x9d means an organic radical of the formula Rxe2x80x94C(O)xe2x80x94, derived from an organic acid by the removal of the hydroxyl group; R is alkyl or aryl of 1-12 carbon atoms; e.g., CH3COxe2x80x94 is the acyl radical of acetic acid (CH3COOH), or acetyl. Another examples for acyl is propionyl. Benzoyl is the acyl radical of benzoic acid (C6H6COOH), etc.
xe2x80x9cLower acylxe2x80x9d refers to xe2x80x9cacylxe2x80x9d when it represents xe2x80x9calkanoylxe2x80x9d which is the organic radical RCOxe2x80x94 in which R is an alkyl group of 1-6 carbon atoms; preferred lower acyl groups are the acetyl and the propionyl radicals.
xe2x80x9cLower alkyloxyxe2x80x9d, xe2x80x9c(lower alkyl)aminoxe2x80x9d, xe2x80x9cdi(lower alkyl)aminoxe2x80x9d, xe2x80x9c(lower alkanoyl)aminoxe2x80x9d, and similar terms mean alkoxy, alkylamino, dialkylamino, alkanoylamino, etc. in which the or each alkyl radical is a xe2x80x9clower alkylxe2x80x9d as described above.
xe2x80x9cHalogenxe2x80x9d or xe2x80x9chaloxe2x80x9d means fluorine, chlorine, bromine, or iodine.
xe2x80x9cTritylxe2x80x9d means the triphenylmethyl radical (PH)3Cxe2x80x94.
xe2x80x9cMesylxe2x80x9d means the methanesulfonyl radical CH3SO2xe2x80x94.
xe2x80x9cTosylxe2x80x9d means the p-toluenesulfonyl radical CH3C6H5SO2xe2x80x94.
xe2x80x9cAprotic (nonpolar) solventxe2x80x9d means organic solvents such as diethyl ether, ligroin, pentane, hexane, cyclohexane, heptane, octane, benzene, toluene, dioxane, tetrahydrofuran, carbon tetrachloride, and the like.
xe2x80x9cPhase transfer catalystxe2x80x9d means a catalyst which alters the rate of transfer of water-soluble reactant across the interface to the organic phase. Suitable catalysts are, e.g., tetrabutylammonium chloride, tetrabutylammonium bromide, tetrabutylphosphonium chloride, tetrabutylphosphonium bromide, and N-2-ethylhexyl-4-dimethylamino pyridinium bromide
xe2x80x9cDerivativexe2x80x9d of a compound means a compound obtainable from the original compound by a simple chemical process.
xe2x80x9cActivated derivativexe2x80x9d of a compound means a reactive form of the original compound which renders the compound active in a desired chemical reaction, in which the original compound is only moderately reactive or non-reactive. Activation is achieved by formation of a derivative or a chemical grouping within the molecule with a higher free energy content than that of the original compound, which renders the activated form more susceptible to react with another reagent. In the context of the present invention activation of the carboxy group is of particular importance and corresponding activating agents or groupings which activate the carboxy group are described in more detail below. An example of an activated derivative of L-valine is the compound of Formula (VI): 
wherein p2 is an amino-protecting group and A is a carboxy-activating group, for example, halo, a lower acyloxy group, a carbodiimide group such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC), an isobutyrate group, and the like.
Of particular interest for the present invention is an amino acid anhydride which is an activated form of an amino acid which renders the amino acid (especially L-valine) susceptible to esterification. Amino acid anhydrides are included in the compounds of Formula (VI), above. Especially useful for the present invention are the cyclic amino acid anhydrides of L-valine, described in PCT Patent Application WO 94/29311, such as 2-oxa-4-aza-5-isopropyl-cycloalkane-1,3-dione of Formula (VIa): 
in which p2 is an amino protecting group. Other examples of the cyclic amino acid anhydrides are protected amino acid N-carboxyanhydrides (NCA""s) described in more detail below.
xe2x80x9cProtecting groupxe2x80x9d means a chemical group that (a) preserves a reactive group from participating in an undesirable chemical reaction; and (b) can be easily removed after protection of the reactive group is no longer required. For example, the benzyl group is a protecting group for a primary hydroxyl function.
xe2x80x9cAmino-protecting groupxe2x80x9d means a protecting group that preserves a reactive amino group that otherwise would be modified by certain chemical reactions. The definition includes the formyl group or lower alkanoyl groups with 2 to 4 carbon atoms, in particular the acetyl or propionyl group, the trityl or substituted trityl groups such as the monomethoxytrityl group, dimethoxytrityl groups such as the 4,4-dimethoxytrityl, the trichloroacetyl group, the trifluoroacetyl group, the silyl group, the phthalyl group, and the N-(9-fluorenylmethoxycarbonyl) or xe2x80x9cFMOCxe2x80x9d group, the allyloxycarbonyl group, or other protecting groups derived from halocarbonates such as (C6-C12)aryl lower alkyl carbonates (such as the N-benzyloxycarbonyl group derived from benzylchlorocarbonate), or derived from biphenylalkyl halocarbonates, or tertiary alkyl halocarbonates such as tertiary butylhalocarbonates, in particular tertiary butylchlorocarbonate, or di(lower)alkyldicarbonates, in particular di(t-butyl)dicarbonate, and the triphenylmethyl halides such as triphenylmethyl chloride, and trifluoroacetic anhydride.
xe2x80x9cHydroxy-protecting groupxe2x80x9d means a protecting group that preserves a hydroxy group that otherwise would be modified by certain chemical reactions. In the context of the present invention, the hydroxy-protecting group can be an ether- or ester-forming group that can be removed easily after completion of all other reaction steps, such as a lower acyl group (e.g., the acetyl or propionyl group), or an aralkyl group (e.g., the benzyl group, optionally substituted at the phenyl ring).
xe2x80x9cSilylation catalystxe2x80x9d as used herein refers to catalysts that promote the silylation of guanine, for example ammonium sulfate, p-toluenesulfonic acid, trifluoromethane sulfonic acid, trimethylsilyltrifluoromethane sulfonate, bistrimethylsilyl sulfonate, sulfuric acid, potassium butylsulfonate, ammonium perchlorate, sodium perchlorate, sodium borofluoride or tin tetrachloride.
xe2x80x9cSilylating agentxe2x80x9d as used herein refers to a compound capable of silylating guanine. A preferred silylating agent is hexamethyldisilazane (which will give a compound of Formula (II)) in which R5, R6, and R7 are all methyl). However, many other silylating agents are known in the art. For example, guanine may be reacted with a trialkylsilyl halide of formula SiR5R6R7X, in which R5, R6, and R7 are independently lower alkyl and X is chloro or bromo, such as trimethylsilyl chloride, tert-butyldimethylsilyl chloride, and the like, preferably in the presence of about 1-2 molar equivalents of a base.
The silylated/persilylated compound of Formula (II) is represented as follows: 
Formula (II) represents guanine protected by one, two, or three silyl groups, or a mixture thereof, where Z1, Z2, and Z3 are independently hydrogen or a silyl group of formula SiR5R6R7, provided that at least one of Z1, Z2, and Z3 must be a silyl group, in which R5, R6, and R7 are independently lower alkyl. It should be noted that Formula (II) as drawn represents a mixture of N-7 and N-9 isomers (as a tautomeric mixture).
Optionally, Formula (II) may represent the case where Z1 is an amino-protecting group other than silyl as defined in the specification, and Z2 and Z3 are independently hydrogen or silyl.
xe2x80x9cLeaving groupxe2x80x9d means a labile group that is replaced in a chemical reaction by another group. Examples of leaving groups are halogen, the optionally substituted benzyloxy group, the mesyloxy group, the tosyloxy group or the acyloxy group.
All the activating and protecting agents employed in the preparation of the compound of Formula (I) must meet the following qualifications: (1) their introduction should proceed quantitatively and without racemization of the L-valine component; (2) the protecting group present during the desired reaction should be stable to the reaction conditions to be employed; and (3) the group must be readily removable under conditions in which the ester bond is stable and under which racemization of the L-valine component of the ester does not occur.
The process of the invention may also include the optical resolution of a prodrug of Formula (I). Terminology relating to the stereochemistry and optical resolution of these compounds is described in European Patent Application 0 694 547 A, incorporated herein by reference.
xe2x80x9cOptionalxe2x80x9d or xe2x80x9coptionallyxe2x80x9d means that a described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances in which it does not. For example, xe2x80x9coptionally substituted phenylxe2x80x9d means that the phenyl may or may not be substituted and that the description includes both unsubstituted phenyl and phenyl wherein there is substitution; xe2x80x9coptionally followed by converting the free base to the acid addition saltxe2x80x9d means that said conversion may or may not be carried out in order for the process described to fall within the invention, and the invention includes those processes wherein the free base is converted to the acid addition salt and those processes in which it is not.
xe2x80x9cPharmaceutically acceptablexe2x80x9d means that which is useful in preparing a pharmaceutical composition that is generally safe and non-toxic and includes that which is acceptable for veterinary use as well as human pharmaceutical use.
xe2x80x9cPharmaceutically acceptable saltsxe2x80x9d means salts which possess the desired pharmacological activity and which are neither biologically nor otherwise undesirable. Such salts include acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or with organic acids such as acetic acid, propionic acid, hexanoic acid, heptanoic acid, cyclopentane-propionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, o-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, p-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, p-toluenesulfonic acid, camphorsulfonic acid, 4-methyl-bicyclo[2.2.2]oct-2-ene-1-carboxylic acid, glucoheptonic acid, 4,4xe2x80x2-methylene-bis-(3-hydroxy-2-naphthoic)acid, 3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acids, salicylic acid, stearic acid, muconic acid, and the like.
Preferred pharmaceutically acceptable salts are those formed with hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzene-sulfonic acid, p-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, p-toluenesulfonic acid and camphorsulfonic acid.
Unless specified to the contrary, the reactions described herein take place at atmospheric pressure within a temperature range from 5xc2x0 C. to 170xc2x0 C. (preferably from 10xc2x0 C. to 50xc2x0 C.; most preferably at xe2x80x9cgroomxe2x80x9d or xe2x80x9cambientxe2x80x9d temperature, i.e., about 20xc2x0 to 30xc2x0 C). However, there are clearly some reactions where the temperature range used in the chemical reaction will be above or below these temperature ranges. Further, unless otherwise specified, the reaction times and conditions are intended to be approximate, e.g., taking place at about atmospheric pressure within a temperature range of about 5xc2x0 C. to about 100xc2x0 C. (preferably from about 10xc2x0 C. to about 50xc2x0 C.; most preferably about 20xc2x0-30xc2x0 C.) over a period of about 1 to about 100 hours (preferably about 5 to 60 hours). Parameters given in the Examples are intended to be specific, not approximate. Isolation and purification of the compounds and intermediates described herein can be effected, if desired, by any suitable separation or purification procedure such as, for example, filtration, extraction, crystallization, column chromatography, thin-layer chromatography or thick-layer chromatography, or a combination of these procedures. Specific illustrations of suitable separation and isolation procedures can be had by reference to the examples hereinbelow. However, other equivalent separation or isolation procedures can, of course, also be used.
While the broadest definition of this invention is set forth in the Summary of the Invention as a process for preparing the compound of Formula (I) and its pharmaceutically acceptable salts, the (R,S) mixture and certain salts are preferred.
The following acids are preferred to form pharmaceutically acceptable salts with the compound of Formula (I): hydrochloric, sulfuric, phoshoric, acetic, methanesulfonic, ethanesulfonic, 1,2-ethanedisulfonic, 2-hydroxyethanesulfonic, benzenesulfonic, p-chlorobenzenesulfonic, 2-naphthalenesulfonic, p-toluenesulfonic and camphorsulfonic acid. Most preferred are strong inorganic acids such as hydrochloric, sulfuric or phosphoric acid.
The most preferred compounds are 2-(2-amino-1,6-dihydro-6-oxo-purin-9-yl)methoxy-3-hydroxy-1-propyl-L-valinate hydrochloride and acetate. These compounds can be prepared as crystalline materials and therefore can be easily manufactured into stable oral formulations.
In any of the last step processes described herein, a reference to Formulae (I), (II), (III), (IV), (V), (VI), (VIa) or (VII) refers to such Formulae wherein Z1, Z2, Z3, and p2, A, Y1, Y2, Z and X are as defined in their broadest definitions set forth in the Summary of the Invention, with the processes applying particularly to the presently preferred embodiments.
The process of the present invention is depicted in the Reaction Sequence shown below: 
wherein Z1, Z2, and Z3 are independently hydrogen or a silyl group of the formula xe2x80x94SiR5R6R7, provided that at least one of Z1, Z2, and Z3 must be a silyl group; optionally, Z1 may be an amino-protecting group selected from the group consisting of lower alkanoyl, optionally substituted trityl, trifluoroacetyl and N-(9-fluorenylmethoxycarbonyl.
The compounds of Formula (III) are glycerol derivatives wherein Y1 and Y2 independently are halo, lower acyloxy, or aralkyloxy, or one of Y1 or Y2 is a valyloxy group, and Z is a leaving group selected from lower acyloxy, benzoyloxy, halo, mesyloxy or tosyloxy, and the like. In general, Y1 and Y2 of the glycerol derivative need to be chosen in such a way as to permit the obtention of the mono-L-valine ester of Formula (I). One of Y1 or Y2 can be an amino-protected L-valyloxy group, or a group convertible to the L-valyloxy group.
The guanine compound of Formula (II) is condensed with a 2-substituted glycerol of the Formula (III) to yield a compound of Formula (IV), which is a 2-(2-amino-1,6-dihydro-6-oxo-purin-9-yl) methoxy-1,3-propanediol (ganciclovir) intermediate with protection at both hydroxy functions (or protection at one hydroxy function when one of Y1 or Y2 is a valyloxy group) and optionally at the 2-amino group of guanine.
When both hydroxy functions are protected, the compound of Formula (IV) is then de-protected at one of the hydroxy functions to provide the mono-protected ganciclovir intermediate of Formula (V).
Optionally, a pharmaceutically acceptable acid addition salt of the compound of Formula (V) may be prepared. Preferred is the hydrochloride acid addition salt of Formula (V).
Compounds of Formula (V) may be esterified with an activated derivative of L-valine of Formula (VI) or (VIa) to provide the compounds of Formulae (VII), optionally followed by removal of amino- and/or hydroxy-protecting groups to form a compound of Formula (I)
If the valyloxy group is introduced in Step (a) using a glycerol derivative where one of Y1 and Y2 is an amino-protected L-valyloxy group or a group convertible to the L-valyloxy group, the resulting compound of Formula (IV) or (V) is converted directly to a compound of Formula (I) by removal of the hydroxy- and amino-protecting groups.
Compounds of Formula (I) can optionally be converted into a pharmaceutically acceptable salt thereof. The process can also include the conversion of an acid addition salt of the prodrug of Formula (I) into a non-salt form, the optical resolution of a compound of Formula (I) or the preparation of the compound of Formula (I) in crystalline form.
The present invention is an improved process for the preparation of mono-L-valine ganciclovir, in which the formation of the intermediate of Formula (V) provides distinct advantages over the previously known procedures. This novel intermediate may optionally be converted into an acid addition salt of a mono-hydroxy protected ganciclovir, which provides for a substantial reduction in several impurities which may be present in the commercially available guanine used to prepare the guanine derivative of Formula (II). These impurities may otherwise be carried through to the desired end-product.
In addition, the starting material for the preparation of some of the glycerol reagents of Formula (III) can be contaminated by certain impurities. These impurities are not removed during synthesis of the glycerol reagent, and when the reagent is reacted with guanine in the condensation reaction, it will give rise to the corresponding isomeric ganciclovir impurities. For example, the starting material for the glycerol reagent of Formula (III), wherein Y1 is benzyloxy and Y2 and Z are propionyloxy, can be the compound 1-benzyloxy-3-chloro-2-propanol. This starting material can contain 2-chloro-3-benzyloxypropanol or 2-benzyloxy-3-chloropropanol. Either of these impurities will give the corresponding impurity in the glycerol reagent and, in the ensuing condensation reaction with guanine, the impurity will carry through as an isomeric impurity of the ganciclovir intermediate.
Furthermore, the reaction of guanine with the glycerol reagent of Formula (III) gives a mixture of product isomers: the desired 9-substituted guanine (the 9-isomer) and a small amount of the undesired 7-substituted guanine (the 7-isomer). If the glycerol reagent contains the impurities discussed above, then the corresponding impurities of ganciclovir will also be present. None of these impurities can be removed easily from the desired 9-isomer.
The present invention provides for the optional generation of an acid addition salt of the compounds of Formula (IV) or (V), which allows for the isolation of the end-product essentially free of the 7-isomer and with levels of the impurities reduced by at least 50%. This acid addition salt intermediate can be prepared directly from the guanine reaction mixture which contains the dihydroxy-protected compound of Formula (IV). Alternatively, and in a preferred embodiment, the compound of Formula (IV) can be first deprotected at one of the hydroxy groups to provide the mono-hydroxy protected ganciclovir of Formula (V), from which intermediate the acid addition salt may then be prepared. Also, from the compound of Formula (IV), one can first prepare the intermediate with protection at both hydroxy moieties and at the 2-amino moiety of the guanine group with, for example, an acyl anhydride. This procedure is advantageous because the fully protected intermediate can be crystallized free of the undesired 7-isomer. These fully protected compounds are novel intermediates and are those compounds of the general Formula (IV), wherein Z1 is an amino-protecting group which is lower acyl, Y1 is halo, lower acyloxy or aralkyloxy, and Y2 is lower acyloxy, so that the acyl groups of Z1 and Y2 are the same. A preferred, fully-protected intermediate is dipropionylmonobenzyl ganciclovir or diacetyl-monobenzyl ganciclovir.
In general, the process for producing the compounds of Formula (I) may or may not involve protection of the amino group in the 2-position of the guanine base. These protecting groups may be removed prior to the formation of the salt intermediate of Formula (V), after the esterification step or in the last deprotection step. For the case when the ganciclovir intermediates have a protected 2-amino group the protecting group may be removed by conventional procedures. For example, if the amino-protecting group is a lower alkanoyl group, basic conditions (pH between 8 to 11) are employed to remove the protecting group. For example, a 2-N-acetylganciclovir ganciclovir intermediate is treated with an alkaline reagent such as ammonium hydroxide, sodium or potassium carbonate or sodium or potassium hydroxide until the removal of the acetyl group is complete. In general, this reaction will be conducted in the presence of a suitable solvent such as a lower alkanol. Preferably the starting material is dissolved in methanol and a stoichiometric excess of ammonium hydroxide is added. The reaction temperature is kept between 0xc2x0 to 50xc2x0 C., preferably at room temperature. After the reaction is complete (which can be determined by TLC), another solvent may be added to facilitate isolation of the de-protected product, such as ethyl ether which leads to precipitation of the de-acylated product which can be filtered off and isolated using conventional separation methods.
In general, when carrying out a process of this invention, those amino, hydroxy or carboxylic groups which are not to participate in the synthesis reaction must be protected until (1) either de-protection yields the final product; or (2) the presence of the unprotected group in the ensuing reaction steps leading to the final product would not modify the intended sequence of reactions. An example for meeting requirement (1) is the benzyloxycarbonyl group in the preparation of the final product of this invention, which protects the amino group of the valine function of ganciclovir until it is removed in the de-protection step. An example for meeting requirement (2) is the acetyl group, or the trityl or monomethoxytrityl group protecting the amino group of the guanine ring system of ganciclovir, as the unprotected amino group does not interfere with the esterification (Step c).
In general, the qualification of potential blocking agents that render them suitable for use in the preparation of the compound of Formula (I) include:
(1) Their introduction should proceed quantitatively and smoothly without L-valine racemization;
(2) The blocked intermediate must be stable to conditions of the reactions employed until removal of the protecting group is required;
(3) The blocking group must be susceptible of being readily removed under conditions which do not change the chemical nature of the remainder of the molecule or result in racemization of the L-valine component. 
where Z1, Z2 and Z3 are independently hydrogen or a silyl protecting group of the formula R5R6R7Si, in which R5, R6, and R7 are independently lower alkyl, provided that at least one of Z1, Z2 and Z3 is a silyl group.
Preparation of Silylated/Persilylated Guanine of Formula (II)
The trialkylsilyl halides of formula R5R6R7SiX (where X is chloro or bromo) or hexamethyldisilazane are commercially available.
As illustrated in Reaction Scheme B, guanine is silylated to give the corresponding protected compound of Formula (II).
The protection of guanine is well known in the art (see, for example xe2x80x9cSynthesis of 9-substituted Guanines. A Reviewxe2x80x9d by F. P. Clausen and J. J. Christensen, Org. Prep. Proced. Int., 25 (4), pp 375-401 (1993)). Guanine may, for example, be protected using acyl groups, for example acetyl, or by silyl groups. Traditionally, when silyl groups are employed for protection, guanine is silylated in such a manner that all active protons present in guanine are replaced by a silyl group before proceeding with the desired reaction, i.e. guanine is protected as the trisilyl derivative. However, it has been found that, although trisilylation of guanine followed by the condensation of Step (a) gives the desired product in good yield, and indeed is preferred, it is not essential that guanine be trisilylated for the condensation carried out in Step (a) to be essentially specific for the preparation of compound (IV). Conventionally, guanine as a slurry is reacted with a silylating agent, for example hexamethyldisilazane, at reflux until all suspended material goes into solution, which signals the complete formation of the trisilyl derivative. This reaction can take up to 48 hours or more. It has been found that refluxing for much less time, for example as little as 2 hours, then reacting the slurry thus produced with a compound of Formula (III) as described in Step (a), gives good yields of desired product. This result is clearly advantageous, since less expense is involved in a shortened reaction time, and smaller amounts of silylating reagent are used. Although the composition of a compound of Formula (II) produced by reacting guanine with hexamethyldisilazane for a shortened period of time is not yet known with any certainty, it is believed to be mainly a monosilyl derivative, probably mixed with some disilyl and trisilyl guanine.
In one preferred method, guanine is reacted with about 3-10 molar equivalents of a silylating agent, preferably with hexamethyldisilazane (i.e. to give a compound of Formula (II) where R5, R6, and R7 are all methyl), in the presence of an silylation catalyst, preferably ammonium sulfate, trifluoromethanesulfonic acid, trimethylsilyltrifluoromethane sulfonate, or bistrimethylsilyl sulfonate, most preferably trifluoromethanesulfonic acid (about 0.01 to 0.1 molar equivalents). The mixture is heated to reflux over a period of about 5-24 hours, preferably about 16 hours. When the reaction is substantially complete, excess silylating agent is removed under reduced pressure, and the resultant solution of the protected guanine product of Formula (II) is used in the next step without further purification.
Alternatively, guanine is reacted with a silylating agent, preferably hexamethyldisilazane, in the presence of a silylating catalyst, preferably trifluoromethanesulfonic acid, as described in the preceding paragraph, but for a period of about 1-8 hours, preferably 2-4 hours. Optionally, excess silylating agent is removed under reduced pressure, and the resultant mixture of the protected guanine product of Formula (II) is used in the next step without further purification.
Alternatively, guanine may be reacted with 1-5 molar equivalents of a trialkylsilyl halide of formula SiR5R6R7X, in which R5, R6, and R7 are independently lower alkyl and X is chloro or bromo, such as trimethylsilyl chloride, tert-butyldimethylsilyl chloride, and the like, in the presence of about 1-5 molar equivalents of a base.
It should be noted that ammonium sulfate, trifluoromethanesulfonic acid, trimethylsilyltrifluoromethane sulfonate, or bistrimethylsilyl sulfonate work well as silylation catalysts in the silylation of guanine described above. However, use of trifluoromethanesulfonic acid is preferred because it is much less expensive than trimethylsilyltrifluoromethane sulfonate or bistrimethylsilyl sulfonate.
Starting Materials
All starting materials employed to make the compound of Formula (I) are known, such as guanine and the protecting and carboxylic-group-activating reagents.
The glycerol derivatives of Formula (III) which are used in the condensation reaction with guanine or a protected guanine compound are described in European Patent Applications 0 694 547 A and 0 187 297. European Patent Application 0 187 297 also describes certain methods for preparing the glycerol derivatives of Formula (III). A preferred method for preparing the glycerol derivatives is described below in the section xe2x80x9cPreparation of Glycerol Derivativesxe2x80x9d.
A preferred guanine derivative is the silylated/persilylated guanine. Preferred glycerol derivatives are 1-benzyloxy-3-propionyloxy-2-(propionyloxy)methoxypropane, 1-benzyloxy-3-acetyloxy-2-(acetyloxy)methoxypropane, or 1-benzyloxy-3-benzyloxy-2-(acetyloxy)methoxypropane.
Prior to carrying out Step (c) (esterification step), the amino group of the L-valine derivative must be protected to avoid its interference with the esterification by undesirable amide formation. The various amino-protected L-valine derivatives useful in this invention, such as N-benzyloxycarbonyl-L-valine, BOC-L-valine and FMOC-L-valine, N-formyl-L-valine and N-benzyloxycarbonyl-N-carboxy-L-valine anhydride, are all commercially available (SNPE Inc., Princeton, N.J., Aldrich Chemical Co., Milwaukee, Wis., and Sigma Chemical Co., St. Louis, Mo.), or are described in the literature, such as N-allyloxycarbonyl-L-valine. Cyclic amino-protected L-valine derivatives are also described in the literature, as noted above. Of particular interest for the present invention is the benzyloxycarbonyl valine-substituted 2-oxa-4-aza-cycloalkane-1,3-dione (Z-valine-N-carboxyanhydride, or Z-Valine-NCA), which is also commercially available (SNPE Inc., Princeton, N.J.). Alternatively, the protecting step may be carried out by conventional methods.
Preparation of Glycerol Derivatives of Formula (III)
The glycerol derivatives useful in this invention can be prepared from known starting materials. For example, the compounds of Formula (III) wherein Y1 is lower aralkyloxy or halo, Y2 is lower acyloxy or halo, and Z is lower acyloxy, can be prepared as described below. This reaction is exemplified by the preparation of the compounds wherein Y1 is benzyloxy, Y2 is propionyloxy and Z is propionyloxy, i.e., 1-benzyloxy-3-propionyloxy-2-(propionyloxy)methoxypropane.
Epichlorohydrin is reacted with benzyl alcohol in the presence of tetrabutylammonium bisulfate in aqueous sodium hydroxide, at room temperature. The product of this reaction, benzyl glycidyl ether, is isolated by conventional means and is then added slowly to a suspension of lithium chloride in tetrahydrofuran and acetic acid, at 40xc2x0-70xc2x0 C., preferably below 60xc2x0 C. The reaction mixture is allowed to cool to room temperature, and stirred for 2-10 hours, preferably 3-6 hours. The product is isolated by extraction, washed and dried to provide 1-benzyloxy-3-chloro-2-propanol. To this product is then added methoxymethyl propionate, which is prepared by adding propionic anhydride to dimethoxymethane in the presence of an ion exchange resin, e.g., Amberlyst 15, maintaining the temperature between 40xc2x0-60xc2x0 C., preferably between 40xc2x0-50xc2x0 C. during the addition. The reaction mixture is aged and cooled, then filtered, washed and distilled. This product, methoxymethyl propionate, is reacted with 1-benzyloxy-3-chloro-2-propanol in an aprotic solvent, e.g., hexanes, in the presence of p-toluenesulfonic acid hydrate at reflux. Distillation and washing affords the product 1-benzyloxy-3-chloro-2-(propionyloxy)methoxypropane. Finally, to prepare the compounds of Formula (III), 1-benzyloxy-3-chloro-2-(propionyloxy)methoxypropane, is refluxed with an alkali metal alkanoate, e.g., sodium propionate, in an aprotic solvent, e.g., toluene, after which a phase transfer catalyst such as tetrabutylphosphonium chloride is added. The reaction mixture is stirred at 90xc2x0 C. to reflux temperature for 1-3 days, preferably 2 days, at which time more tetrabutylphosphonium chloride and solvent may be added, if required. The mixture is heated to reflux and the distillate removed, then stirred at 90xc2x0 C. to reflux temperature for 3-16 hours, preferably 5-10 hours, then cooled to ambient temperature. The mixture is then washed with water and brine, and the organic phase is separated and concentrated to yield 1-benzyloxy-3-propionyloxy-2-(propionyloxy)methoxy propane. In an analogous manner, other glycerol derivatives of Formula (III) may be prepared.
Further nonlimiting examples of phase transfer catalysts or agents that may be employed in the preparation of compounds of Formula (III) are reviewed by C. M. Starks, C. L. Liotta, and M. Halpern in xe2x80x9cPhase-Transfer Catalysisxe2x80x9d, Chapman and Hall, New York, 1994, which is incorporated herein in its entirety by reference.
Preparation of Activated derivative of L-valine
Prior to carrying out Step (c) (esterification step), L-valine must also be activated. At least 1 equivalent of the protected amino acid and 1 equivalent of a suitable coupling agent or dehydrating agent, for example 1,3-dicyclohexylcarbodiimide or salts of such diimides with basic groups should be employed from the start. Other carbodiimides such as N,Nxe2x80x2-carbonyldiimidazole may also be used. Further useful dehydrating agents are trifluoroacetic anhydride, mixed anhydrides, acid chlorides, 1-benzo-triazolyloxy-tris-(dimethylamino)phosphonium hexafluorophosphate, benzotriazol-1-yl-oxy-trispyrrolidinophosphonium hexafluorophosphate, 1-hydroxybenzotriazole, 1-hydroxy-4-azabenzotriazole, 1-hydroxy-7-azabenzotriazole, N-ethyl-Nxe2x80x2-(3-(dimethylamino)propyl)carbodiimide hydrochloride, 3-hydroxy-3,4-dihydro-4-oxo-1,2,3-benzotriazine, O-(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate, O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate, O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate, O-(1H-benzotriazol-1-yl)-1,1,3,3-bis(tetramethylene)uronium hexafluorophosphate or O-(7-azabenzotriazol-1-yl)-1,1,3,3-bis-(tetramethylene)uronium hexafluorophosphate.
A description of these coupling agents by L. A. Carpino can be found in J. Am. Chem. Soc. 1993, 115, p. 4397-4398.
Also useful for this purpose are urethane-protected amino acid N-carboxy anhydrides (UNCA""s) which are an activated form of an amino acid; these have been described by William D. Fuller et al., J. Am. Chem. Soc. 1990, 112, 7414-7416, which is incorporated herein by reference. Other protected amino acid N-carboxy anhydrides are described in PCT Patent Application WO 94/29311, discussed above. In summary, any other reagent that produces an anhydride or another activated derivative of the protected amino acid under mild conditions can be used as the coupling agent.
The amino-protected amino acid is dissolved in an inert solvent such as a halogenated lower alkane, preferably dichloromethane, under an inert atmosphere, for example nitrogen, and the coupling agent is added (preferably 1,3-dicyclohexylcarbodiimide). The reaction mixture is stirred at temperatures between 0xc2x0 and 50xc2x0 C., preferably at about room temperature. The reaction mixture is filtered and the reaction product (the anhydride of the protected amino acid) isolated. The resulting product is dissolved in a dry inert solvent such as dry dichloromethane and placed under nitrogen.
Preparation of Mono-L-valine Ganciclovir
Step (a): Condensation
The reaction conditions for the condensation of guanine with the 2-amino group optionally protected, are described in European Patent Application 0 187 297. In this condensation reaction, guanine is reacted with a glycerol derivative of Formula (III) in an aprotic hydrocarbon solvent (such as benzene, toluene or xylenes) or dimethylformamide with a hexa-lower alkyl(di)silazane, for example, hexamethyldisilazane, hexaethyldisilazane, or the like, and a catalyst at temperatures between 30xc2x0 C. and reflux temperature. The catalyst is a Lewis acid salt such as trialkyl silyl salt (e.g., the sulfate), or a trifluoroalkyl sulfonic acid, a chlorosilane, or ammonium sulfate and pyridine. For a more detailed disclosure of the reaction conditions for condensation (Step (a)) see the disclosure of European Patent Application 0 187 297 which is incorporated herein by reference. The resulting compound is a ganciclovir derivative with protected hydroxy groups and with an optionally protected 2-amino group.
For example, a ganciclovir intermediate of Formula (IV), where Y1 is benzyloxy and Y2 is lower acyloxy, can be prepared by condensing persilyl guanine with a glycerol derivative of Formula (III) where Y1 is benzyloxy and Y2 and Z are lower acyloxy. Typically, persilyl guanine is treated with a large excess of a glycerol derivative of Formula (III) in the presence of a catalytic amount of a Lewis acid salt, preferably trifluoromethane sulfonic acid at 60xc2x0-150xc2x0 C. preferably 110xc2x0-130xc2x0 C. for 3-24 hours, preferably 6-8 hours. The mixture is cooled, diluted with an aprotic nonpolar solvent, preferably toluene, and then water is added carefully. The product can optionally be isolated by filtration.
Step (b): Hydrolysis
The protected ganciclovir derivative of Formula (IV) from Step (a) is partially de-protected to provide ganciclovir with the 2-amino group optionally in protected form and one protected primary hydroxyl function. Preferably, the primary hydroxyl function is protected with a benzyl group. Suitable amino-protecting groups are lower alkanoyl groups with 2 to 4 carbon atoms, in particular the acetyl or propionyl group. Other suitable amino-protecting groups are the trityl or substituted trityl groups such as the monomethoxytrityl group, and the 4,4xe2x80x2-dimethoxytrityl group.
As noted above, the acid addition salt of the compound of Formula (V), can be prepared directly from the product of Step (a), which is the dihydroxy-protected compound of Formula (IV), by de-protecting one of the hydroxy groups with concomitant preparation of the salt. Alternatively, the compound of Formula (IV) can first be deprotected at one of the hydroxy groups to provide the mono-hydroxy protected ganciclovir of Formula (V), from which the acid addition salt is then prepared. Also, the compound of Formula (IV), the intermediate with protection at both hydroxy groups as well as at the 2-amino guanine group can be prepared, with, for example, an acyl anhydride. For example, the dipropionyl monobenzyl ganciclovir intermediate is prepared from the propionyl monobenzyl ganciclovir intermediate of Formula (IV) by reaction with propionic anhydride/dimethylamlnopyridine, in, for example, toluene. As discussed above, the ganciclovir intermediate with protection at both hydroxy groups and at the 2-amino guanine group, such as dipropionyl monobenzyl ganciclovir, is a preferred intermediate because it can be isolated substantially free of the undesired 7-isomer of guanine.
When one of Y1 and Y2 is aralkyloxy, or when both Y1 and Y2 are aralkyloxy, for example, benzyloxy, then deprotection occurs by hydrogenolysis under conventional hydrogenation conditions; when one of the groups Y1 or Y2 is acyloxy or halo, said group is selectively removed by basic hydrolysis.
Transfer hydrogenation conditions can also be employed: a palladium catalyst such as palladium hydroxide is used in a suitable solvent such as cyclohexene. A cosolvent such as methanol, ethanol or isopropanol may be necessary for better solubility of the adduct.
Hydrogenolysis is preferably carried out by dissolving the protected ganciclovir in a solvent system under conventional hydrogenation conditions at 5-100 psi (0.3-7 atm), preferably 10-40 psi (0.7-2.8 atm) hydrogen, in the presence of a catalyst such as a palladium compound, in particular palladium hydroxide on carbon (Pearlman""s catalyst), at about 20xc2x0-60xc2x0 C., preferably 20xc2x0-35xc2x0 C., until completion of the reaction. Other suitable hydrogenation catalysts include hydrogenation catalysts in general such as Pd, Pd on carbon and homogeneous hydrogenation catalysts. The solvent system includes a lower alkanol such as methanol or ethanol. Generally the reaction will be carried out at temperatures between room temperature and the reflux temperature of the solvent system, for example in refluxing ethanol under a hydrogen atmosphere and under exclusion of air. The reaction vessel is preferably swept with nitrogen prior to charging it with hydrogen. The catalyst will be recovered by filtration. The filtrate can be reduced in volume by evaporation of excess solvent. The resulting crude reaction mixture generally includes unchanged starting material and 2-amino-protected ganciclovir with one aliphatic hydroxy group protected as the major products. The separation of these two products is usually performed by isolation procedures known in the art, often by chromatographic methods, preferably on silica gel, followed by elution with appropriate eluents such as mixtures of a lower alkanol with a halogenated lower alkane (preferably ethanol and dichloromethane) to give 2-amino-protected ganciclovir with one aliphatic hydroxy group protected. This ganciclovir intermediate can then be isolated as the salt compound of Formula (V) by conventional methods, using, for example, hydrogen chloride and a solvent such as methanol.
The hydrolysis reaction to remove an acyl hydroxy-protecting group is preferably carried out by treating the protected ganciclovir under basic hydrolysis conditions. The hydrolysis medium may include a lower alkyl alcohol such as methanol or ethanol, toluene, and aqueous sodium hydroxide. Generally the reaction will be carried out at temperatures between room temperature and the reflux temperature of the solvent system. Again, this ganciclovir intermediate can be isolated as the salt compound of Formula (V) as described above.
For example, the product obtained in Step (a) can be partially deprotected by removing the lower acyl group (of Y1 or Y2) with base. After the reaction described in Step (a) is complete and the reaction mixture has been cooled and diluted with, preferably, methanol, aqueous sodium hydroxide is added. The mixture is heated to 40xc2x0-90xc2x0 C., preferably 60xc2x0-80xc2x0 C., until the reaction is complete. The reaction mixture is then carefully acidified with hydrochloric acid. The product is collected as the hydrochloride salt by filtration, then washed and dried.
Step (c): Esterification
In this step an activated derivative of amino-protected L-valine of the Formula (VI) or (VIa) is esterified with the mono-hydroxy protected ganciclovir salt derivative of Formula (V) obtained in Step (b). Suitable amino-protecting groups for the L-valine derivative are the N-benzyloxycarbonyl group, the phthalyl group, the tertiary butyloxycarbonyl group and the N-(9-fluorenylmethoxycarbonyl) or xe2x80x9cFMOCxe2x80x9d group.
A suspension of the product of Step (b) (the compound of Formula (V) in an aprotic solvent (preferably dimethylformamide) containing an organic base (preferably TEA) is added to an approximately equivalent amount of the activated L-valine derivative in an aprotic solvent (preferably dimethylformamide). The activated L-valine derivative is preferably Z-valine-N-carboxyanhydride or L-valine anhydride. The reaction mixture is stirred at 0xc2x0-40xc2x0 C., preferably at 4xc2x0-10xc2x0 C., for 1-5 hours. The reaction mixture is diluted with water, preferably toluene and water. The precipitate is collected by filtration, washed and dried at ambient temperature.
Step (d): Final De-protection to Give the Product of Formula (I)
The valine protecting groups of the product of Step (c), the hydroxy protecting group Y2 and optionally any 2-amino guanine protecting groups are removed by de-protection reactions, preferably in an acidic medium or solvent, most preferably by hydrogenation. De-protection under acidic conditions is preferred, as this will ensure that the amino group liberated in the de-protection reaction will be protonated; that is, that the base of Formula (I) as it is formed in the de-protection reaction will be captured by an at least stoichiometric amount of acid present. Isolating the compound of Formula (I) as an acid addition salt will protect the desired stereoconfiguration of the compound of Formula (I). Therefore, those examples given below that show the de-protection step also show the concomitant salt formation step.
The de-protection reaction is carried out by dissolving the product of the esterification step (c) in an inert solvent, preferably in an acidic solvent, using a hydrogenation catalyst such as palladium on carbon, or palladium hydroxide on carbon (Pearlman""s catalyst), using elevated hydrogen pressure between 1 and 2000 psi (0.1-140 atm), preferably 20 to 200 psi (1.4-14 atm). The completion of the reaction can be monitored using conventional TLC analysis. The hydrogenolysis is continued until the conversion is complete, if required with addition of further hydrogenation catalyst. The catalyst is removed and washed. The combined filtrates from filtration and the washings are concentrated and lyophilized to isolate the ganciclovir L-valine ester. The purification of the product and the isolation of a crystalline ester is carried out by recrystallization or other purification techniques such as liquid chromatographic techniques.
The hydrogenolysis may be slow due to the presence of impurities (catalyst poisons) in the starting material. It has been found to be advantageous to treat the starting material prior to hydrogenolysis in methanol with filtering aids such as catalytic Filtrol(copyright), Solka Floc(copyright) and activated carbon such as ADP carbon. This effectively removes most catalyst poisons.
If the tertiary butyloxycarbonyl group is being used as amino-protecting group, its removal is effected with acid such as HCl and isopropanol as a solvent or with trifluoroacetic acid neat.
Alternatively, if the esterification step has been carried out with a trityl or substituted trityl-protected ganciclovir derivative, such protecting groups can be removed by treatment with an aqueous alkanoic acid or trifluoroacetic or hydrochloric acid at temperatures between xe2x88x9220xc2x0 C. and 100xc2x0 C., for example, aqueous acetic acid.
Preparation of Salts
One of ordinary skill in the art will also recognize that the compound of Formula (I) may be prepared either as an acid addition salt or as the corresponding free base. If prepared as an acid addition salt, the compound can be converted to the free base by treatment with a suitable base such as ammonium hydroxide solution, sodium hydroxide, potassium hydroxide or the like. However, it is important to point out that the free base of Formula (I) is more difficult to characterize than its acid addition salts. When converting the free base to an acid addition salt, the compound is reacted with a suitable organic or inorganic acid (described earlier). These reactions are effected by treatment with an at least stoichiometric amount of an appropriate acid (in case of the preparation of an acid addition salt) or base (in case of liberation of the free compound of Formula (I)). In the salt-forming step of this invention typically, the free base is dissolved in a polar solvent such as water or a lower alkanol (preferably isopropanol) or mixtures thereof, and the acid is added in the required amount in water or in lower alkanol. The reaction temperature is usually kept at about 0xc2x0 to 50xc2x0 C., preferably at about room temperature. The corresponding salt precipitates spontaneously or can be brought out of the solution by the addition of a less polar solvent such as ether or hexane, removal of the solvent by evaporation or under vacuum, or by cooling the solution.
Isolation of Stereoisomers and the Manufacture of Crystalline 2-(2-Amino-1,6-dihydro-6-oxo-purin-9-yl)methoxy-3-hydroxy-1-propyl-L-valinate
From the Formula (I) it is apparent that the compound of the invention has one asymmetric carbon atom (chiral center) in the propyl chain, in addition to the asymmetric carbon atom in L-valine. Therefore, two diastereomeric forms exist, the (R)- and (S)-form as determined by the rules of Cahn et al. Suitable methods for the separation of the diastereomers are described in European Patent Application 0 694 547 A, incorporated herein by reference.
The compounds of Formula (I) may also be prepared in crystalline form, which has many well-known advantages over the non-crystalline form. Suitable methods for the preparation of the compounds of the invention in crystalline form are also described in European Patent Application 0 694 547 A, incorporated herein by reference.